The Prime Minister said he had come across many employers who think it would be "crazy" to embark on border controls and different currencies and urged them to voice their concerns.
Speaking in the Commons during Prime Minister's Questions, Mr Cameron said: "A huge amount of pressure is put on businesses by the Scottish Government with all sorts of threats and warnings if they speak out and say what they believe is the truth.
"I come across business leader after business leader, large and small, in Scotland that want to keep our UK together, who think it would be crazy to have border controls, different currencies and split up our successful UK.
"I would urge them to speak out, talk with their work forces about the strength of our UK and vote to keep it together."
He was responding to a question from Labour's Jim Sheridan (Paisley and Renfrewshire North) who insisted employers have a "moral responsibility" to inform employees of any consequences of separation.
The MP told the House: "The uncertainty surrounding the future of Scotland and indeed the UK has resulted in many of the British community in Scotland withholding significant investments in that country.
"Do you therefore agree with me there is a moral responsibility on employers to inform their employees what the consequences are, if any, of separation of Scotland from the UK in order they can make an informed choice prior to the referendum?"